DESCRIPTION: Cancer cells frequently inactivate DNA double strand break (DSB) repair proteins causing genomic instability and the genetic evolution of tumors. Inactivation of these proteins usually does not cause overt defects in DNA DSB repair due to the activity of compensatory repair pathways. We reason that identifying compounds that cripple these compensatory pathways will be the first step in developing therapeutic agents that can sensitize tumor cells, but not normal tissues to genotoxic cancer therapies. The DNA damage response histone protein H2AX is a dosage-dependent tumor suppressor that exhibits reduced expression in many cancers. H2AX deficiency leads to genomic instability, but not to overt defects in DNA DSB repair by non-homologous end joining (NHEJ). Deficiency in the NHEJ factor XLF also does not lead to demonstrable defects in NHEJ. However, the combined deficiency of H2AX and XLF leads to a severe block in DSB repair by NHEJ. These findings demonstrate that H2AX and XLF are in compensatory DSB repair pathways and that inhibition of one pathway in cells deficient in the other will lead to a profound block in NHEJ. In conjunction with the University of New Mexico Center for Molecular Discover (UNMCMD), we have developed a flow cytometric high throughput screen for chemical compounds that inhibit NHEJ. This screen has been optimized and used to screen a small library of compounds (1,500) achieving Z' scores of >0.6. Here, we propose to use this approach to screen a larger, more complex chemical library to identify compounds that inhibit NHEJ specifically in H2AX-deficient, but not wild type cells. We refer to these compounds as H2AX-specific Synthetic Inhibitors of NHEJ (SINs). A linear, multistep approach to validate the function of these compounds will also be developed. We believe that developing agents that selectively inhibit NHEJ in cancer cells will ultimately enable us to exploit DNA repair pathway defects to treat cancers. Moreover, this approach has significant advantages over current efforts to selectively inhibit the other major pathway of DSB repair, homologous recombination (HR) in cancer cells. Whereas HR only functions in DSB repair in dividing cells, NHEJ is required for DSB repair in all cells in the G0-G1 phases of the cell cycle. Thus, agents identified by our screen, which inhibit NHEJ will be effective in treating all cancer cells including the vast majority that are not actively dividing. Importantly, cancer stem cells, which are thought to be the basis for relapse of many cancers after treatment, exist primarily in G1 and would also be sensitized by SINs. In addition to identifying H2AX-specific SINs, completion of these studies will establish a linear pipeline for th future identification of SINs for treating cancers defective in a broad range of DNA DSB repair proteins. Moreover, they will also identify novel DSB repair pathways used by normal cells and cancer cells.